Treatment following hamstring strain injury (HSI) is complicated by a lack of prognostic indicators and high rates of re-injury. Spatial frequency (SF) analysis, a quantitative ultrasound method to assess structural tissue organization, could complement clinical evaluations. PURPOSE: To characterize differences in SF parameters between injured and uninjured limbs following acute HSI in Division I collegiate athletes. METHODS: Ultrasound imaging was performed within one week of HSI, also confirmed by MRI. Longitudinal B-mode images (Aixplorer, Supersonic Imagine, Aix-en-Provence, France) were acquired by a single musculoskeletal-trained sonographer using a linear array transducer (2-10 MHz) at the injury site of maximum tenderness and corresponding location for the uninjured limb. A region of interest (ROI) was drawn about the site of injury and location-matched on the images of the uninjured limb. A 2D Fourier Transform was performed on all possible 5 mm square kernels within the ROI. Peak spatial frequency radius (PSFR), a parameter corresponding to the frequency of the fascicular banding pattern, and Mmax%, a ratio comparing the prominent banded pattern relative to the background, were computed for each kernel. PSFR and Mmax% from all kernels were averaged across respective ROIs. Paired t-tests were used to compare parameter differences between limbs. RESULTS: Seven male athletes sustained HSI (mean age = 19.1 ± 0.8 yrs). PSFR (1/mm) was lower (p = 0.005) in the injured limb (0.81 ± 0.22) compared to the uninjured limb (0.95 ± 0.18). Mmax% was also lower (p = 0.023) in the injured limb (1.44 ± 0.41) compared to the uninjured limb (2.03 ± 0.56). Both measures indicated decreased tissue organization in the injured limb, characterized by disruption of the reflected fascicular banded pattern. CONCLUSION: SF analysis successfully detected differences between injured and uninjured muscle tissue. Although differences in SF parameters between limbs cannot differentiate whether alterations are due to mechanical disruption of fascicles or presence of edema, this method may have promise in identifying structural changes following HSI and in monitoring changes throughout recovery.Supported by NBA & GE Healthcare Orthopedics and Sports Medicine Collaboration and NIH Grant UL1TR002373 and TL1TR002375.